Phosphatase proteins, particularly members of the Cdc14 phosphatase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to develop improved Cdc14 nucleic acid constructs for improved expression and purification of Cdc14 proteins. The present invention advances the state of the art by providing novel Cdc14 nucleic acid constructs/vectors and methods of using such constructs/vectors for expressing and purifying modified Cdc14 phosphatase proteins which are expressed as a single form at a high yield and can be readily purified to homogeneity.
Protein Phosphatases
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. The biochemical pathways through which signals are transmitted within cells comprise a circuitry of directly or functionally connected interactive proteins. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of certain residues on proteins. The phosphorylation state of a protein may affect its conformation and/or enzymic activity as well as its cellular location. The phosphorylation state of a protein is modified through the reciprocal actions of protein kinases (PKs) and protein phosphatases (PPs) at various specific amino acid residues.
The protein phosphorylation/dephosphorylation cycle is one of the major regulatory mechanisms employed by eukaryotic cells to control cellular activities. It is estimated that more than 10% of the proteins active in a typical mammalian cell are phosphorylated. The high-energy phosphate that confers activation and is transferred from adenosine triphosphate molecules to proteins by protein kinases is subsequently removed from the proteins by protein phosphatases. In this way, the phosphatases control most cellular signaling events that regulate cell growth and differentiation, cell-to-cell contacts, the cell cycle, and oncogenesis.
Three protein phosphatase families have been identified as evolutionarily distinct. These include the serine/threonine phosphatases, the protein tyrosine phosphatases, and the acid/alkaline phosphatases (Carbonneau H. and Tonks N. K. (1992) Annu. Rev. Cell Biol. 8:463–93).
The serine/threonine phosphatases are either cytosolic or associated with a receptor. On the basis of their sensitivity to two thermostable proteins, inhibitors 1 and 2, and their divalent cation requirements, the serine/threonine phosphatases can be separated into four distinct groups, PP-I, PP-IIA, PP-IIB, and PP-IIC.
PP-I dephosphorylates many of the proteins phosphorylated by cylic AMP-dependent protein kinase and is therefore an important regulator of many cyclic AMP mediated, hormone responses in cells. PP-IIA has broad specificity for control of cell cycle, growth and proliferation, and DNA replication and is the main phosphatase responsible for reversing the phosphorylations of serine/threonine kinases. PP-IIB, or calcineurin (Cn), is a Ca.sup.+2-activated phosphatase; it is involved in the regulation of such diverse cellular functions as ion channel regulation, neuronal transmission, gene transcription, muscle glycogen metabolism, and lymphocyte activation.
PP-IIC is a Mg.sup.++-dependent phosphatase which participates in a wide variety of functions including regulating cyclic AMP-activated protein-kinase activity, Ca.sup.++-dependent signal transduction, tRNA splicing, and signal transmission related to heat shock responses. PP-IIC is a monomeric protein with a molecular mass of about 40–45 kDa. One alpha. and several beta. isoforms of PP-IIC have been identified (Wenk, J. et al. (1992) FEBS Lett. 297: 135–138; Terasawa, T. et al. (1993) Arch. Biochem. Biophys. 307: 342–349; and Kato, S. et al. (1995) Arch. Biochem. Biophys. 318: 387–393).
The levels of protein phosphorylation required for normal cell growth and differentiation at any time are achieved through the coordinated action of PKs and PPS. Depending on the cellular context, these two types of enzymes may either antagonize or cooperate with each other during signal transduction. An imbalance between these enzymes may impair normal cell functions leading to metabolic disorders and cellular transformation.
For example, insulin binding to the insulin receptor, which is a PTK, triggers a variety of metabolic and growth promoting effects such as glucose transport, biosynthesis of glycogen and fats, DNA synthesis, cell division and differentiation. Diabetes mellitus, which is characterized by insufficient or a lack of insulin signal transduction, can be caused by any abnormality at any step along the insulin signaling pathway. (Olefsky, 1988, in “Cecil Textbook of Medicine,” 18th Ed., 2:1360–81).
It is also well known, for example, that the overexpression of PTKs, such as HER2, can play a decisive role in the development of cancer (Slamon et al., 1987, Science 235:77–82) and that antibodies capable of blocking the activity of this enzyme can abrogate tumor growth (Drebin et al., 1988, Oncogene 2:387–394). Blocking the signal transduction capability of tyrosine phosphatases such as Flk-1 and the PDGF receptor have been shown to block tumor growth in animal models (Millauer et al., 1994, Nature 367:577; Ueno et al., Science, 252:844–848).
Relatively less is known with respect to the direct role of phosphatases in signal transduction; PPs may play a role in human diseases. For example, ectopic expression of RPTP.alpha. produces a transformed phenotype in embryonic fibroblasts (Zheng et al., Nature 359:336–339), and overexpression of RPTP.alpha. in embryonal carcinoma cells causes the cells to differentiate into a cell type with neuronal phenotype (den Hertog et al., EMBO J 12:3789–3798). The gene for human RPTP.gamma. has been localized to chromosome 3p21 which is a segment frequently altered in renal and small lung carcinoma. Mutations may occur in the extracellular segment of RPTP.gamma. which renders a RPTP that no longer respond to external signals (LaForgia et al., Wary et al., 1993, Cancer Res 52:478–482). Mutations in the gene encoding PTP1C (also known as HCP, SHP) are the cause of the moth-eaten phenotype in mice that suffer severe immunodeficiency, and systemic autoimmune disease accompanied by hyperproliferation of macrophages (Schultz et al., 1993, Cell 73:1445–1454). PTP1D (also known as Syp or PTP2C) has been shown to bind through SH2 domains to sites of phosphorylation in PDGFR, EGFR and insulin receptor substrate 1 (IRS-1). Reducing the activity of PTP1D by microinjection of anti-PTP1D antibody has been shown to block insulin or EGF-induced mitogenesis (Xiao et al., 1994, J Biol Chem 269:21244–21248).
Cdc14 Phosphatases
Cdc14 is a member of the dual-specificity protein tyrosine phosphatase family, and is related to PTEN/MMAC1 (also known as “phosphatase and tensin homolog”, “phosphatase and tensin homolog deleted on chromosome 10”, and “mutated in multiple advanced cancers 1”) (Li et al., J. Biol. Chem. 272 (47), 29403–29406 (1997)). The human Cdc14 protein is similar to the yeast (Saccaromyces cerevisiae) Cdc14 protein, which has been shown to play important roles in the exit of cell mitosis and initiation of DNA replication and is crucial for cell cycle progression in Saccaromyces cerevisiae. The yeast Cdc14 phosphatase regulates the cell cycle by dephosphorylating proteins that have been phosphorylated by the cyclin-dependent kinase Cdc28/clb. In humans, Cdc14 phosphatases have been shown to interact with and dephosphorylate the tumor suppressor protein p53, suggesting that Cdc14 is important in humans for controlling the cell cycle and regulating the function of p53 (Li et al., J. Biol. Chem. 275 (4), 2410–2414 (2000)). The human Cdc14A gene has been mapped to human chromosome band 1p21, which is a chromosomal region that has been shown to exhibit loss of heterozygosity in highly differentiated breast carcinoma and malignant mesothelioma (Wong et al., Genomics 59 (2), 248–251 (1999)). A 48 bp deletion in the Cdc14A gene has been identified in a breast carcinoma cell line, and loss of expression of the wild-type allele in the breast cancer cell line indicates that Cdc14A may be a tumor suppressor gene that is inactivated during tumorigenesis (Wong et al., Genomics 59 (2), 248–251 (1999)).
Because of these important biological functions, Cdc14 phosphatase proteins, and encoding nucleic acid molecules, are well established in the art as having valuable commercial utilities related to cancer and other disorders, such as for developing agents for the prognosis, diagnosis, prevention, and/or treatment of cancer.
The amino acid sequence of the art-known Cdc14, homolog A, isoform 1 protein (referred to herein as “Cdc14A1”) is disclosed in Genbank gi:15451929, and the nucleotide sequence of the art-known Cdc14A1 mRNA transcript is disclosed in Genbank gi:15419128. These art-known Cdc14A1 protein and encoding nucleic acid sequences are also disclosed in U.S. Pat. No. 6,331,614 (Wong et al., issued Dec. 18, 2001). References herein to the “native” Cdc14A1 gene/protein are intended to refer to genes/proteins having these art-known sequences.
Further information on the Cdc14 gene/protein, particularly the Cdc14A1 variant, can be found in the following patent and journal articles: U.S. Pat. No. 6,331,614 (Wong et al., issued Dec. 18, 2001); Wong et al., “Genomic structure, chromosomal location, and mutation analysis of the human CDC14A gene”, Genomics 59 (2), 248–251 (1999); Li et al., “A family of putative tumor suppressors is structurally and functionally conserved in humans and yeast”, J. Biol. Chem. 272 (47), 29403–29406 (1997); Li et al., “The human Cdc14 phosphatases interact with and dephosphorylate the tumor suppressor protein p53”, J. Biol. Chem. 275 (4), 2410–2414 (2000); and OMIM entry No. 601728 (for information on PTEN/MMAC1).
Expression and Purification of Phosphatase Proteins
Although it is well recognized in the art that phosphatase proteins, particularly Cdc14 proteins, have valuable commercial and medical utilities due to the important roles that they play in such biological functions as cell cycle regulation and tumorigenesis, it is also recognized that expression and purification of phosphatase proteins such as Cdc14 is problematic. This has hindered the usefulness of phosphatase genes/proteins in the development of therapeutic agents, such as for treating cancer. For example, expression of a phosphatase gene may lead to the production of numerous forms of the phosphatase protein having different molecular weights, and it may be difficult to determine which, if any, of these forms are active phosphatases. Various other impurities may also be co-expressed along with the intended phosphatase protein. For example, the GST portion of a fusion protein may be expressed by itself.
Thus, a need exists in the art for modified phosphatase proteins, and nucleic acid constructs for expressing these proteins, that can be efficiently expressed as a single form of the intended phosphatase with high yield and that can be readily purified to homogeneity, while still retaining the phosphatase activity of the native phosphatase enzyme.
Consequently, the development of modified Cdc14 proteins and nucleic acid expression constructs, and methods of making and using them for improved expression and purification of Cdc14 phosphatase proteins, satisfies a need in the art by providing new compositions and methods that are useful in the development of human therapeutic and diagnostic agents, particularly for cancer.